A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions

Klöhn, Peter-Christian; Stoltze, Lars; Flechsig, Eckhard; Enari, Masato; Weissmann, Charles

doi:10.1073/pnas.1834432100

Cited by 302 publications

(442 citation statements)

References 25 publications

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“…They fall into three categories: Western blots, enzyme-linked immunosorbent assays (ELISAs), and the conformation dependent immunoassay (CDI). The most widely used test is the Western blot, which is reported to detect between 10 to 20 pmol ofÅ PrP ScÅ [16Å -18].Å ELISAÅ methodologyÅ isÅ moreÅ sensitive, andÅcanÅdetectÅ2ÅpmolÅofÅPrP ScÅ [19Å -21].ÅTheÅCDI immunoassay is the most sensitive method reported, capable of detection of 0.1 pmol of PrP Sc in brain homogenateÅ (2Å ngÅ ofÅ PrPÅ ofÅ MWÅ 16,241)Å [22].Å Methods to amplify prion titer before detection using either cell cultureÅ [23]Å orÅ cell-freeÅ conversionÅ assaysÅ [24,Å 25]Å show considerable promise. Despite the advancements made in analytical methodology for prion analysis, apparently the detection limit needs to be improved by about three orders of magnitude to allow detection of prions in blood (vide supra, 30 amol/mL in blood versus 0.1 pmol detection by the best antibody methods).…”

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confidence: 99%

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Onisko¹,

Dynin²,

Requena

et al. 2007

J. Am. Soc. Mass Spectrom.

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Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein.

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mentioning

confidence: 99%

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Onisko¹,

Dynin²,

Requena

et al. 2007

J. Am. Soc. Mass Spectrom.

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“…However, there is also evidence that prion multiplication show cell tropism ex vivo. This is best illustrated for the ME7 strain which does not replicate in N2a or GT1 cell lines [16,67] but does replicate in SN56 [7], L929 [147], and MG20 [61] cell lines.…”

Section: Fibroblast-likementioning

confidence: 99%

“…Following the observation that only a small percentage of cells are actually infected in a typical N2a culture population [115], N2a subclones with much higher permissiveness were isolated [16,41]. These N2a sublines, along with improved detection of PrP res , allowed the development of a quantitative, highly sensitive cell-based infectivity assay for the RML murine prion strain [67]. In the scrapie cell assay (SCA), N2a cells are exposed for a few days to the infectious inoculum, split three times at a 1:10 dilution, then spotted onto filters.…”

Section: Cell Models As Sensitive Bioassay For Prion Infectivitymentioning

confidence: 99%

Cell models of prion infection

Vilette

2007

Vet. Res.

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-Due to recent renewal of interest and concerns in prion diseases, a number of cell systems permissive to prion multiplication have been generated in the last years. These include established cell lines, neuronal stem cells and primary neuronal cultures. While most of these models are permissive to experimental, mouse-adapted strains of prions, the propagation of natural field isolates from sheep scrapie and chronic wasting disease has been recently achieved. These models have improved our knowledge on the molecular and cellular events controlling the conversion of the PrP C protein into abnormal isoforms and on the cell-to-cell spreading of prions. Infected cultured cells will also facilitate investigations on the molecular basis of strain identity and on the mechanisms that lead to neurodegeneration. The ongoing development of new cell models with improved characteristics will certainly be useful for a number of unanswered critical issues in the prion field.

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“…[7][8][9] Some recent approaches for ante mortem diagnosis include applying newly developed ligands against PrP sc , 10,11 conformational-dependent immunoassays, 12,13 spectroscopic techniques, 14,15 and PrP sc amplification. 16,17 Efforts to avoid protease digestion and use of other means to differentiate PrP sc and PrP c are the most desirable, but these methods are under trial and no practical application has been suggested so far. Fischer et al reported the use of plasminogen as a ligand that interacts specifically with PrP sc and not with PrP c .…”

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Prion Protein Detection Using Nanomechanical Resonator Arrays and Secondary Mass Labeling

et al. 2008

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Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 µg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 µg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.Prion proteins (PrP) are transmissible infectious particles, devoid of nucleic acid, that cause fatal neurodegenerative diseases known as bovine spongiform encephalopathy (BSE) in bovine, scrapie (SC) in sheep, and Creutzfeldt-Jakob disease (CJD) in humans. 1 These diseases are caused by genetic, infectious, or sporadic disorders where host-encoded, noninfectious cellular prion proteins (PrP c ) are converted into a conformationally altered, infectious forms designated as PrP sc , PrP CJD , and PrP BSE . 2,3 PrP c is present in abundance in most tissues of the central nervous system. However, posttranslational processes convert PrP c into PrP sc , which leads to altered physiochemical and biochemical properties such as aggregation, insolubility, protease digestion resistance, and a -sheet-rich secondary structure.One such altered property of PrP sc , namely, protease digestion resistance, forms the basis of several diagnostic biochemical tests.To differentiate between PrP c and PrP sc , the sample is pretreated with protease K. Since PrP sc is digestion resistant and PrP c is easily digested by protease K, pretreatment results in a sample that is rich in PrP sc as compared to PrP c . 4,5 Because neither the sensitivity of PrP c nor the resistance of PrP sc to digestion is absolute, the "protease sensitivity assay" cannot definitively measure the presence or absence of PrP.Current prion detection methods employ post mortem analysis after suspicious animals manifest one or more symptoms of the disease. These post mortem tests include gel electrophoresis and western blot, direct-binding and sandwich ELISA, and conformational-dependent immunoassay. [4][5][6] To improve food safety it would be beneficial to screen all the animals for prion disease using ante mortem testing, regardless of the presence of symptoms. Because ante mortem tests could be performed on presymptomatic animals, they would therefore be required to detect extremely small amounts of PrP circulating in blood samples and would have to differentiate PrP c and PP ...

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A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions

Cited by 302 publications

References 25 publications

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Mass spectrometric detection of attomole amounts of the prion protein by nanoLC/MS/MS

Cell models of prion infection

Prion Protein Detection Using Nanomechanical Resonator Arrays and Secondary Mass Labeling

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